Acquity system

The presence of 10% water in the eluent minimized variations. Two different LC modes were used for the analysis of the extracts a smaller (50 x 2.1 mm) Atlantis C18 column (5 /um particle size) on a Shimadzu liquid chromatograph and a UPLC column (Acquity C18,1.7 fim particle size, 50 x 2.1 mm) on a Waters Acquity system. In the Shimadzu experiment, a gradient from 0.5 min... 

Nordstrom et al. [121] also compared the differences between UPLC and HPLC for metabolomic profiling. This time a Waters Acquity system was coupled to Micromass Q-Tof-Micro mass spectrometer and data were acquired in the m/z 100-1000 range with an acquisition of 2 spectra per second. About 20% more components were detected using UPLC versus HPLC and the length of the chromatographic separation was one of the most crucial parameters affecting the number of detected features. These examples demonstrate the importance of appropriate mass analyzers when utilizing UPLC. For more on UPLC see Chapter 8. 

Waters Ultra Performance Liquid Chromatography Acquity System, Waters Corp., Milford, MA, 2004. 

Figure 5.5 Plots of extra-column variance versus mobile phase flow rate. Instruments Waters Acquity UPLC, Agilent 1200, Agilent 1100, Shimadzu Nexera, Peridn Elmer Flexar, Merck Hitachi LaChrom. The possible maximum acquisition rate was set on each instrument (10 Hz on Merck Hitachi LaChrom system, 80 Hz on Agilent 1100 and 1200 systems, and 100 Hz on Shimadzu Nexera, Perkin-Elmer Flexar, and Waters Acquity systems). From Fekete, S., Fekete, J. J. Chromatogr. A. 2011, with permission.

Based on Table 3.2, we can deduce from the available flow rates that some manufacturers clearly optimized their systems for 2.1 mm inner diameter (ID) columns, namely the Jasco X-LC, the Thermo Accela, and the Waters Acquity. By sacrificing flexibility in column ID, these systems have been completely optimized to these 2.1 mm columns. Achieving that will be explained below. The instruments of Agilent and Dionex mentioned in Table 3.2 involve a more flexible approach. The higher flow rate limits of their systems allow the use of columns with larger Ids. Users benefit from the better efficiency obtained with 4.6 or 3.0 mm ID columns instead of 2.1 mm ID columns of similar length and their systems are fully compatible with existing conventional methods. 

A Waters Acquity UPLC system with a cooling autosampler and column oven was used. The stationary phase was a Waters Acquity BEH C18 column (50 x 2.1 mm, 1.7 /.un particle size). The column was maintained at 40°C. The mobile phase consisted of water and acetonitrile, each containing 0.3% formic acid and was delivered at 0.35 mL/min in a gradient mode at 60% water from 0 to 1.5 min, linearly decreased to 10% water in 0.5 min, and then returned to 60% water. Sample vials were maintained at 4°C. 

Lercanidipine — Kalovidouris et al.57 applied UPLC-MS/MS to the determination of lercani-dipine in human plasma after oral administration of lercanidipine. A Waters Acquity UPLC system with cooling autosampler and column oven was coupled with a Waters BEH C18 column (50 x 2.1 mm, 1.7 jum). The mobile phase was composed of 70% acetonitrile in water containing 0.2% v/v formic acid, delivered at a flow of 0.30 mL/min. The column temperature was maintained at 40°C and sample vials at 5°C. 

A commercial HPLC system and columns capable of performing ultra high-pressure LC were recendy introduced at PITTCON 2004 (ACQUITY Ultra Performance LC System by Waters). This HPLC system was designed to take full advantage of the potential of novel, sub-2-micron particles to give scientists chromatographic run times that are up to 9 times shorter than current fast HPLC systems, up to 2 times better peak capacity or resolution, and 3 times better routine sensitivity. 

FIGURE 5 (a) Peptide digest run on a 4.8-gm particle on a traditional HPLC system. Peak count is 70, and peak capacity is 143. (b) Peptide digest run on a 1.7-gm particle on the ACQUITY UPLC system. Peak count is 168, and peak capacity is 360. (Courtesy of Waters Corp.)... 

Figure 1.2. (a) The traditional technique of low-pressure liquid chromatography using a glass column and gravity-fed solvent with manual fraction collection, (b) A modern automated HPLC instrument (Waters Acquity UPLC system) capable of very high efficiency and pressure up to 15,000 psi. 

Figure 4.21. Effect of instrumental dispersion on gradient analysis on a short 2-mm column, (a) Analysis on a standard HPLC system, (b) Analysis of the same sample on a low-dispersion system (Waters Acquity). Diagram courtesy of Waters Corporation.

In order to overcome the pressure limitations and the other challenges. Waters Corporation introduced Acquity UPLC , the first commercially available system that addresses the challenge of using elevated pressure and sub-2 pm particles, which makes it a particularly attractive and promising analysis tool [51]. The UPLC instrumentation is designed to deliver mobile phase at pressures up to 1034 bar (15,000 psi). The researchers were very active in this area for some time to manufacture columns that can withstand these rigorous pressures [52,53]. 

The system was equipped with a trifunctional high-strength silica (100% silica particle) analytical column (Acquity HSS T3 100 x 2.1 mm i.d., 1.8 pm particle size), using an isocratic mobile phase composed of aqueous 0.1% (v/v) formic acid at a flow rate of 250 pL/min. The injection volume was 20 pL, while the detection wavelength for the PDA was set at 245 nm, and the analytical colunm was kept at room temperature [75]. 

Reversed-phase UPLC separation system was also used for the determination of four water-soluble B vitamins, including B5, B8, B9, and B12 in fortified infant foods using Waters Acquity UPLC BEH C,g column (100 x 2.1 mm i.d., 1.7 pm particle size), and a binary gradient, acetonitrile-water mobile phase [85]. 

These applications have been reported by using the Acquity triple quadrupole detector (TQD) [28-39], the Micromass Quattro Premier triple quadrupole (QqQ) system [40], the Micromass Q-TOFII [18], and the Q-TOF Ultima [27] from Waters. As can be seen in these tandem MS equipments, two different analyzers have been reported, QqQ [28-40], the same as the two analyzers, and Q-TOF [18,27] as the hybrid system. The QqQ analyzer was the only one reported for the analysis of biological samples [30-40]. Generally, when the QqQ analyzer was used, quantification studies were performed due to their higher sensitivity in the selected ion monitoring (SRM) mode, and when the Q-TOF was used, identification or characterization was done due to its higher power for confirmation purposes. 

UPLC-MS/MS analysis 10 pL of each extract was injected onto a UPLC-ESI-MS/MS system. Chromatographic separations were carried out using the Acquity UPLC BEH (bridged ethyl hybrid) C18 column 2.1 x 100 mm, and 1.7-pm particle size. The column was eluted under gradient from 95% of eluent A and 5% of eluent B to 100% of eluent B over 10.0 min, at a flow rate of 0.3 mL min. Eluent A water/ formic acid (0.1%, v/v) eluent B acetonitrile/formic acid (0.1%, v/v). 

The need for increased separation performance is the driving force behind UPLC. This new technology will fulfill this need either for high-power separations in an acceptable time frame, or for very rapid separations with more conventional resolution, or for any intermediate scenario that can profit from both the increase in performance and the gain in speed. The ACQUITY UPLC system represents the first step into this new direction of chromatography. 

FIGURE 16.8 High efficiency analysis of a test sample using five coupled Acquity 1.7-pm columns at near ambient temperature (40 C). Analysis conditions Total column length, 450 mm flow rate, 0.17 ml/min (system) pressure, 999 bar temperature, 40°C (Polaratherm) detection, 210 nm (20 Hz). Mobile phase 30/70 acetonitrile/water. Peak identification 1 = uracil, 2 = caffeine, 3 — pyridine, 4 — aniline, 5 — phenol, 6 — acetophenone, 7 = benzene. Source Reproduced with permission from 2006 Elsevier B.V. 

There are several software and automated systems for HPLC method development and optimization, such as Drylab , Chromsword , and ACD/AutoChrom MDS, and others (43 7). Their principles can be applied to UHPLC. In addition. Waters Corp. (Milford, MA) has recently promoted Fusion Method Development software. Fusion Method Development software from S-Matrix integrates seamlessly with Water s ACQUITY UPLC and Empower 2 Chromatography software to automate method development. The software automatically generates instrument methods and sample sets. Another feature of this software is to visualize data by statistically fitting the results. However, it cannot generate simulated chromatograms at predicted conditions, like Drylab can. 

For a UHPLC system, a similar procedure can be used. However, the flow rate needs to be adjusted according to maximum pressure of the UHPLC system. In addition, a restrictor may be needed if the back pressure is too low. The typical dwell volume for most UHPLC systems is 0.1 mL as shown in Figure 1.11. The approximate dwell volumes for Waters Acquity and Agilent 1290 are listed in Table 1.6. 

X 2.1 mm column 1 p,L injection 0.2 mg/mL analyte in the mobile phase Acquity UPLC system. 

A new tool designed to meet the challenges which conventional HPLC system encountered became generally available in 2004. In this year, an ACQUITY UPLC system was introduced (18). It is the first commercially available UHPLC system solving... 

---